Biosynthetic Experiments

The aim of biosynthetic experiments with fungal metabolites is to establish the structure of the building blocks, the order in which they are assembled, the way in which chains are folded to form the carbon skeleton and the sequence interrelating precursors with the final metabolite. Biosynthesis is concerned with both sequences and reaction mechanisms. The sequence of the biosynthetic events, the role of intermediates and the stereochemistry of enzymatic reactions can be studied with appropriately isotopically-labelled substrates and with structural analogues of the natural intermediates. The chemical enzymology of individual steps and the role of key components and structures of the enzyme may be studied with isolated enzyme systems obtained from fungi. The features that determine the function of the enzyme and which control its activity may be determined by genetic studies in which mutants play an important role. 

An objection to a biosynthetic result, particularly if the incorporation of the putative precursor is low, is that the possible precursor is being degraded to a 

Deuterium and even 0 can induce a small isotope shift in the position of a C signal and thus C can be used as a reporter nucleus for the presence of these isotopes. The NMR spectrum and the presence of couplings 

Subsequent chapters give many examples of the perceptive and intellectually stimulating use of isotopic labels to discriminate between biosynthetic pathways. 

Unlike primary metabolites, the genes that regulate the formation of the enzymes of seeondary metaboUte biosynthesis are often clustered. In several eases the loei of these genes have been determined. This has considerable sig-nifieanee in the control of secondary metabolite biosynthesis. The genes that eode for several important polyketide pathways such as those leading to the aflatoxins and the statins have been identified. Similar work has also been reported for penicillin biosynthesis and some non-ribosomal peptides as well as terpenoid pathways such as that leading to the gibberellins. 

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No biosynthetic experiments have been reported for these compounds, but they probably all share the same biosynthetic mechanism. One possibility is that they are generated by cyclization of an a-amino-p-keto carboxyl intermediate that would arise from threonine (136) and sphingosine (131) for 139 and 130, respectively (Figure 11.23). Alternatively, cyclization may precede oxidation, with an aziridine intermediate being formed. 

Secondary metabolites generated via the propionate route are quite unusual in nature. Relevant exceptions are some antibiotic macrolides from Streptomycetes [42], but wholly propionate-derived macrolides are rare. This biosynthetic pathway has been well proved for erythromycin (13), where the aglycone is produced by assembling seven propionate units [43, 44], and for a few related antibiotics [45]. However, very sophisticated biosynthetic experiments [46] have established that some apparent propionate units in other macrolides (e.g., aplasmomycin [46]) from Streptomycetes could be formed either by C-methylation through S-adenosylmethionine or from glycerol. 

Pulmonates are air-breathing molluscs that do not include many families and genera [68]. Siphonaria species from many distinct geographical areas possess a series of secondary metabolites with polypropionate skeletons characterized by the presence of ketal, pyrone and furanone moieties [47]. A rigorous biosynthetic experiment [69] proved that Siphonaria denticulata is able to... 

Further biosynthetic experiments were performed with the aim to clarify metabolic relationships between polygodial and the esters, as well as to ascertain whether or not polygodial was the precursor of the esters. Two series of experiments [86] were performed injecting labelled [2-14C] mevalonic acid into nine (4.5 pCi) and fourteen (7 pCi) animals, which were sacrificed at various times from 3 to 77 h. The two experiments, performed during the months of March and April, 1984, led to apparently conflicting but not contradictory results. In fact, in the first series of experiments the major specific recovered radioactivity was always associated with polygodial (30). On the other hand, the... 

Another example of a nudibranch, which probably modifies dietary metabolites to obtain more effective allomones, is seen in Aldisa cooperi (= A. sanguinea cooperi) [155]. It elaborates two fish antifeedant bile acids (104,105) that are absent in its prey, the sponge Anthoarcuata graceae, where the main steroid is 4-cholesten-3-one (106). Biosynthetic experiments starting from both labelled mevalonic acid and labelled 4-eholesten-3-one would definitely clarify, whether, the two allomones (104-105) are biosynthetized de novo by the mollusc, or if they are derived from a food source. 

Interestingly, the first biosynthetic experiments with [3H]linolenic add and the terrestrial plant Senecio isatideus (Asteraceae) as a model system for the biosynthesis of algal pheromones were unsuccessful. If, however, labeled dodeca-3,6,9-trienoic add is administered, a rapid transformation into ectocarpene takes place (36). Nevertheless, the C12 add ultimately derives from linolenic acid via three /3-oxidations, since labeled tetradeca-5,8,11-trienoic acid, which requires only one /3-oxidation, is converted into labeled ectocarpene albeit with very low effidency (37). 

In the course of biosynthetic experiments involving Streptomyces UC5319, Cane and Rossi treated the acidic fraction of an ether extract with diazomethane and obtained 600 which they called pentalenolactone E methyl ester Paquette s solution to the total synthesis of this substance (Scheme LI) was founded upon a... 

Earlier proposals (12, 13, U) and the results of biosynthetic experiments (18) have been adumbrated into a scheme of biosynthesis for the procyanidins (Figure k) in which it is suggested that they are formed as byproducts during the final stage of the synthesis of the parent flavan-3-ol structures, (+)-catechin and (-)-epicatechin (11, 18). A two step reduction of the f1av-3-en-3-ol... 

Regarding these recent results obtained from biosynthetic experiments, one is tempted to say that matters have become rather complicated, despite the considerable amount of new information available. The main question, whether chanoelavine-I (4) is a true intermediate, remains to be solved. 

In most biosynthetic experiments with plants great specificity has been observed throughout the pathway and sequences leading to individual metabolites could be envisaged with a high degree of certainty. Chain shortening of the Cjs triyne skipped-cne 46 was thus found to occur by either... 

No real evidence exists concerning the in vino formation of the carbon-carbon triple bond but dehydrogenation via cis double bonds was favoured speculatively and appears probable on account of the similar incorporations observed for linoleate and crepenynate (5) - and the better incorporations of 14-c( r- than 14-rra is-dehydrocrepenynate (43) into several fungal metabolites (e.g. 35, 36, 40) . Biosynthetic experiments with leaf homogenates of Chrysanthemum f/oscii/osum indicate that the enzymes required for the desaturation of oleic acid are located within the chloroplasts whilst the final oxidation of the C13 triyne skipped-ene... 

For the biosynthetic experiments mentioned a considerable number of potential precursors specifically labelled with and have been synthesized, e.g. References 39-41, 46. 

The design of a biosynthetic experiment depends on whether the putative precursor is to be labelled with a stable ( C, H) or a radioactive ( H, C, isotope, the... 

The sequence of the various steps between uroporphyrinogen III and cobyrinic acid has been the subject of much recent work. A major advance in this area was the observation that sirohydrochlorin (77), the iron-free prosthetic group of the enzyme siroheme, could be modified to accommodate its role as a biosynthetic intermediate. Subsequently a dimethyl isobacteriochlorin (factor II) isolated from P. shermanii was shown to be identical with sirohydrochlorin from E. coli sulphite reductase. The complete stereostructure of (77) was elucidated by a series of biosynthetic experiments using [4- C]- and [5- C]-ALA and independently by more classical structural arguments (B-79MI10401, B-79MI10402>. Sirohydrochlorin labelled biosynthetically from [4- C]ALA and [ CHsJmethionine is incorporated into cobyrinic acid by cell-free extracts of F. shermanii without loss or migration of label. 

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